In the field of clinical laboratory tests, various diseases are diagnosed using biological samples (blood, urine and the like), and as methods for diagnosing them various assay methods have been developed and utilized. Representatives of such assay methods are biochemical assay methods utilizing enzymatic reactions and immunological assay methods utilizing antigen-antibody reactions. As reagents employed in such diagnoses, those relating to a pregnancy test, RA test for detecting a rheumatoid factor, CRP test for detecting a C-reactive protein, as well as tests for hepatitis B surface antigen (HBs antigen), anti-HBs antibody, β2 microglobulin antibody, mycoplasma antigen, nucleic acids, nucleic proteins, estrogen, anti-estrogen antibody and the like have been developed.
As such assay methods, an immunonephelometry (TIA method), latex nephelometry (LIA method), enzyme immunoassay (EIA method) and radioimmunosassay (RIA method) may be exemplified, and are selected appropriately depending on the purpose.
Among those listed above, an LIA method is applied to the detection of various antigens and antibodies since it is convenient and can rapidly be conducted, in which a latex carrier particle formed by dispersing a carrier particle in an aqueous medium is sensitized with an antigen or an antibody, and then used to detect the reaction with a corresponding antibody or antigen in serum as an aggregation reaction of the carrier particle.
A recent trend in medical practices is a change from a conventional diagnosis of a disease to a prophylaxis of a disease. Thus, by means of a test of a blood and the like prior to the onset of a disease, the predisposition of the disease is identified beforehand, whereby effecting the prophylaxis. For the purpose of application of such preventive medical cares, a further higher sensitivity is demanded in performing the LIA method and the like. While an immunoserological test such as an antigen-antibody reaction originally assays a small amount of a substance, a reagent for the assay used in the prophylactic medicine should be capable of detecting a disease-associated trace protein (antigen and/or antibody) at a further lower concentration. Thus, an assay reagent which is even more sensitive than those employed currently becomes essential.
In view of the problems discussed above, an instrument of automatic immunological analysis instrument used for immunological tests to assay a small amount of a sample and a small amount of a reagent has increasingly been improved, and correspondingly raised a demand for a higher sensitivity of the reagent employed in such an instrument.
As a method for increasing the sensitivity of such a reagent, a method is exemplified in which an analyze is attempted to be assayed at a higher sensitivity by increasing the particle size of a carrier particle employed whereby increasing the magnitude of the change of optical density. Japanese Kokoku Publication Sho-58-50645 discloses a method for producing a latex which comprises copolymerizing styrene with a styrene sulfonate in an amount of 10% by weight or less based on said styrene in water using a persulfate as an initiator in the absence of an emulsifier followed by heating under an alkaline condition, and purports that a latex consisting of a carrier particle whose particle size is 0.3 to 0.8 μm can be obtained by increasing the amount of a catalyst based on styrene monomer. On the other hand, Japanese Kokoku Publication Hei-1-36484 discloses a method for producing a diagnostic agent by synthesizing a latex in aqueous solution containing an oxide or hydroxide of a divalent metal.
However, a method employing a latex comprising a carrier particle having a large particle size involves problems that when the analyte present at high concentration, the change of optical density resulting from the carrier particle aggregation exceeds the measurable range to make it impossible to obtain a change of optical density corresponding to the amount of the analyte, that it is likely to reflect a non-specific aggregation reaction and also that the storage over a prolonged period is impossible due to the absence of the stability.
On the other hand, Japanese Kokai Publication Sho-63-65369 discloses a method employing a latex reagent obtained by sensitizing latexes comprising two or more kinds of carrier particles having different average particle sizes with an antibody or antigen and mixing in a certain ratio. This method attempts to achieve a performance having two profiles, namely, a wide range of the assay attributable to the latex comprising a carrier particle having a small particle size and a high sensitivity at a low concentration region attributable to the latex consisting of a carrier particle having a large particle size.
Japanese Kokoku Publication Sho-63-14783 discloses a latex consisting of two kinds of carrier particles having different particle size ranges which are loaded with an identical antigen or antibody in at least two different amounts.
Japanese Patent No. 2588174 discloses a method for assaying an antigen-antibody reaction which comprises reacting a latex obtained by sensitizing two or more kinds of particles having different average particle sizes with an antigen or antibody followed by mixing or a latex obtained by mixing two or more particles having different average particle sizes followed by sensitizing with the antigen or antibody, with an antigen against the sensitizing antibody or an antibody against the sensitizing antigen in water and measuring the change of the absorbance upon irradiation, wherein a carrier particle having an average particle size of 0.05 to 0.3 μm and a carrier particle having an average particle size of 0.3 to 1.0 μm are mixed and wherein a light whose wavelength is at least 2.5 times the average particle size of the mixed particles and is also 0.6 to 2.4 μm are irradiated.
Japanese Kokai Publication Hei-5-18973 discloses an immunological assay method which comprises, depending on the amount of a component to be assayed by an immunological reaction, a carrier particle having a particle size of 0.1 μm or less in which a component reactive with the component to be assayed is insolubilized is combined with at least one of the component reactive with the component to be assayed and a carrier particle having a particle size more than 0.1 μm in which the reactive component is insolubilized, and subjected to the reaction with a sample containing the component to be assayed, as well as a reagent employed in the same.
However, such methods employing the latex consisting of several carrier particles having different average particle sizes are difficult to prepare a latex reagent, and, has a problem that the obtained reagent varies in the performance time by time of the preparation even if an identical operator prepares the reagent in accordance with a certain protocol using the particle having an identical average particle size and an identical CV values.